Project Summary An explosion of recent studies has indicated that altered mechanical forces in the microenvironment of cells, or its ?mechano-some?, is a potentially targetable and quantifiable factor in disease, much like changes in the ge- nome or proteome. Valuable insights into the mechanical microenvironment at the cell and tissue level have been achieved by measuring forces that cells exert on deformable surfaces or their macroscopic stiffness, but have largely ignored how cells sense and respond to force at the molecular level. Changes in macroscopic stiffness in disease are accompanied by a wealth of molecular changes in a cell's tensional homeostasis where ?mechanotransduction? signaling pathways are aberrantly activated. At the epicenter of tension sensing are transmembrane cell-surface receptors, which are uniquely positioned to sense and integrate all cellular me- chanical cues from outside, inside, and within the membrane of the cell. Our overall hypothesis is that studying how cell-surface receptors change conformation to sense and respond to force will lead to a critical under- standing of the mechanical microenvironment of cells at a molecular level thus leading to novel therapeutics and diagnostic tools for many diseases. While advanced single molecule spectroscopy tools exist to probe force-induced conformational changes at a molecular level, decoding mechanotransduction mechanisms has been crippled by a lack of tools to measure how cells sense and respond to force at a molecular level and re- quires synergy between ?cellular-biophysics? and ?structure-function? approaches within the NIGMS mission. To tackle the challenge of measuring molecular-level forces that cells sense in order to identify cell-surface mechanosensors, define magnitudes of physiologic forces, and measure how force changes during disease progression, new hybrid fluorescent molecular tension sensors will be devised that marry advantages of cur- rent genetically-encoded and immobilized DNA-based sensors using a new fusion-tag technology that allows covalent attachment of DNA nanostructures to genetically-encoded proteins in cells. To tackle the challenge of measuring downstream cellular effects of applying force to specific cell-surface receptors, an improved version of a high-throughput magnetic tweezers assay developed to study mechanotransduction of Notch receptors will be used, which applies piconewton forces to magnetic beads tethered to specific receptors, and measures downstream responses using imaging and cell-lysate based readouts such as transcription, localization of adaptor proteins, cytoskeleton dynamics, and relevant kinase and GTPase activity. To tackle the challenge of decoding mechanisms that receptors use to sense and respond to force, x-ray crystallography and an im- proved single molecule proteolysis assay will be used to test the hypothesis that force-induced proteolysis is a general mechanosensing mechanism, as was recently discovered for Notch receptors. By characterizing the cellular ?mechano-some? at a molecular level, these studies have the potential to identify new therapeutic ave- nues and diagnostic tools, and generally elucidate the role of mechanical forces in disease pathogenesis.